The small GTPase Cdc42 is highly conserved from yeast to humans and mediates a number of fundamentally important cellular processes including the establishment of cell polarity, the regulation of cell morphogenesis, and the control of cell growth and differentiation. In past funding periods, we have used a combination of biochemical, cell biological and structural approaches to gain insights into how Cdc42 impacts EGF receptor-signaling and influences cell growth, as well as how it affects cell migration, and cellular differentiation. More recently, we have been combining these approaches with the conditional knock-out of Cdc42 in mice, and the use of small molecules to probe its cellular and biological responses, which has led us to make some surprising new discoveries. In particular, we found that the two highly similar splice-variant forms of Cdc42, one that is ubiquitously distributed and the other that is brain- specific, have distinct functions in neurogenesis, with the ubiquitous form of Cdc42 stimulating the transition of multi-potent embryonic cells to neuroprogenitors, while the brain-specific protein (Cdc42b) is necessary for their differentiation into neurons and glial cells. An important reason for these distinct functions is the unique ability of the ubiquitous form of Cdc42 to activate mTORC1. Moreover, we discovered that Cdc42, through its ability to signal to mTORC1, also regulates glutamine metabolism in the mitochondria, which has significant consequences for the glutamine addiction of cancer cells. Thus, in the coming project period we plan to build on these new findings to address three fundamentally important questions. 1) How is Cdc42 localized at the appropriate cellular sites to propagate signals that are important for cell growth and development? We are especially interested in determining the molecular basis by which Cdc42 versus Cdc42b localize to their specific membrane sites to trigger different signaling outcomes. 2) Define the signaling pathway used by the ubiquitous form of Cdc42 and how it accounts for the functional specificity exhibited by Cdc42 versus Cdc42b. In particular, we will set out to learn how Cdc42 signals to mTORC1 and what is unique about its signaling partners versus those used by Cdc42b to initiate terminal differentiation. 3) How does Cdc42 influence cellular metabolism and thereby impact processes important both to cancer progression and neural development? We want to better understand how Cdc42 works through mTORC1 and NFkB to influence glutamine metabolism and the metabolic re- programming critical for malignant transformation, as well as see whether there are similarities that extend to the role of Cdc42 in the formation of neuroprogenitor cells. These studies should provide important new insights into the roles of Cdc42/Cdc42b in normal cellular and biological processes, as well as how malfunctions in their signaling capabilities can contribute to a variety of disease states and developmental disorders. PUBLIC HEALTH RELEVANCE: The Rho GTPase Cdc42 plays fundamentally important roles in the regulation of cell growth, polarity and differentiation, and thereby impacts a number of developmental processes and disease states. Recently, we have uncovered some potentially exciting new connections between how Cdc42 influences specific steps during neurogenesis and how it signals to the metabolic machinery of cancer cells. By understanding how Cdc42 mediates these outcomes, we hope to shed new light on key events in neural development and developmental disorders as well as in malignant transformation. The expectation is that this information will highlight novel targets and strategies for therapeutic intervention.